Extracellular Biopolymer Production by Acrostalagmus luteoalbusfrom Agro-Industrial Wastes: Toward Sustainable Material Development

Extracellular Biopolymer Production by Acrostalagmus luteoalbusfrom Agro-Industrial Wastes: Toward Sustainable Material Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Extracellular Biopolymer Production by Acrostalagmus luteoalbus from Agro-Industrial Wastes: Toward Sustainable Material Development 1,*Raquel Gómez-Pliego, Judith Espinosa-Raya PhD, Harold Alexis Prada-Ramírez PhD, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7901410/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Plastic pollution has intensified the search for biodegradable alternatives from renewable sources. Microbial fermentation using agro-industrial residues offers a sustainable strategy for producing biopolymers with reduced environmental impact. This study evaluated the production and characterization of an extracellular biopolymer synthesized by Acrostalagmus luteoalbus using low-cost carbon substrates. Methodology: Fermentations were carried out for eleven weeks using pulp with tejocote peel, fruit peels, and sucrose-based media . Biopolymer yields were quantified, followed by physicochemical characterization , elemental analysis (CHNS) , and thermal assessments (TGA and DSC) to evaluate structural and functional properties. Results: All substrates supported biopolymer synthesis, with pulp with tejocote peel yielding the highest production (~17.10% ± 1.29 at week nine), indicating a strong influence of substrate composition and incubation time . The biopolymer was dark brown, brittle, insoluble in polar and non-polar solvents, and thermally stable , with degradation occurring above 250 °C . CHNS analysis showed a carbon-rich, low-nitrogen composition , while TGA and DSC revealed multi-step degradation and no melting transitions , suggesting a heterogeneous, cross-linked polymeric network . Discussion: The extracellular nature simplifies recovery compared to intracellular polymers and combined with thermal stability and solvent resistance , supports applications in biodegradable packaging, coatings, and biomedical materials. Agro-industrial residues represent a cost-effective and sustainable carbon source for biopolymer production. Conclusions: Acrostalagmus luteoalbus offers a promising platform for producing biodegradable, thermally stable biopolymers from agro-industrial wastes, contributing to circular economy strategies and industrial-scale sustainability efforts. Biopolymers Applied & Industrial Microbiology Biopolymer production Agro-industrial waste Acrostalagmus luteoalbus Sustainable materials Waste-to-resource Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The synthesis and widespread use of synthetic polymers revolutionized modern life in the mid-20th century. Due to their versatility, low cost, lightness, and durability, plastics became ubiquitous in packaging, textiles, construction, automotive, and consumer goods [1]. However, these same properties also cause severe environmental problems, since plastics persist for decades or centuries in soils, oceans, and the atmosphere, accumulating as hard-to-manage waste. Global plastic production has increased dramatically from around 2 million tons in 1950 to over 450 million tons in 2023 [2,3]. Annual plastic waste generation now exceeds 350 million tons worldwide [3,4]. Once in the environment, plastics fragment into microplastics that enter food chains and affect ecosystems and potentially human health [5]. Inadequate disposal of plastic waste leads to soil degradation, wildlife mortality through accidental ingestion, and the release of toxic emissions (especially dioxins and furans) during uncontrolled incineration [6,7]. Plastics exhibit extreme persistence, often lasting for centuries in the environment [8,9], highlighting the urgent need for sustainable alternatives. Plastic pollution has become a global crisis, with an estimated 19 to 23 million tons of plastic waste leaking into aquatic ecosystems each year, polluting rivers, lakes, and oceans, and posing severe threats to biodiversity, coastal economies, and human health [10]. In this context, these materials have gained attention as sustainable alternatives to conventional plastics [11]. They consist of macromolecules derived partially or entirely from renewable biological sources (such as plants, algae, and microorganisms) and typically exhibit biodegradability and biocompatibility [12,13]. Bioplastics can be engineered to integrate into circular systems such as reuse, composting, anaerobic digestion, and organic recycling, thereby reducing reliance on fossil resources and helping to close carbon cycles [14,15]. Biodegradation generally involves the breakdown of polymer chains by microorganisms into carbon dioxide, water, and biomass under aerobic conditions, or methane under anaerobic conditions. These biological processes are often complemented by abiotic mechanisms such as hydrolytic, thermal, and photo-oxidative degradation, depending on the polymer’s chemical structure and environmental factors [16,17]. Biopolymers can be broadly classified into four categories: (i) natural renewable polymers such as starch, cellulose, and chitin; (ii) microbial polymers, including polyhydroxyalkanoates (PHAs); (iii) chemically synthesized polymers derived from renewable biomonomers such as polylactic acid (PLA); and (iv) biodegradable petrochemical-based polymers, such as polycaprolactone (PCL) [18,19]. Within these, polyhydroxyalkanoates (PHAs) such as polyhydroxybutyrate (PHB) have been extensively studied as biodegradable thermoplastics, with growing research directed toward low-cost feedstocks like lignocellulosic biomass and agro-industrial residues [20–22]. Despite these advances, industrial-scale PHA production remains limited due to high production costs, low yields, and downstream processing challenges [20,23]. In contrast, filamentous fungi remain underexplored as PHA producers, even though they exhibit strong metabolic adaptability and can utilize complex carbon sources such as agro-wastes and food residues [24]. Given these limitations, exploring alternative microbial taxa with unexplored biosynthetic capabilities is crucial for expanding the range of biopolymer-producing organisms.Recent studies have explored the valorization of agro-industrial residues for microbial biopolymer production. Selvam et al. [25] reviewed the potential of agricultural residues such as sugarcane bagasse, rice husks, banana peels, and spent mushroom substrate for microbial fermentations aimed at biopolymer synthesis. Similarly, Jørgensen et al. [26] analyzed enzymatic approaches for lignocellulose conversion into fermentable sugars, while Barua et al. [27] discussed advances in the sustainable transformation of lignocellulosic wastes into bioplastic precursors. These valorization strategies not only reduce raw material costs but also mitigate the environmental burden associated with waste disposal, reinforcing the transition toward sustainable bioeconomies. Moreover, bioplastics have broad applications, ranging from food packaging, disposable utensils, and textiles to biomedical devices, sutures, drug-delivery systems, and agricultural films [28]. Despite these advances, no microbial biopolymer has yet matched the mechanical or thermal performance of polyethylene, polypropylene, or PET, and challenges remain in improving durability, stability, and lowering costs [28]. The genus Acrostalagmus is recognized for its metabolic versatility and production of diverse metabolites, including antimicrobials and toxins [29]. Acrostalagmus luteoalbus has been identified in damp environments and building materials [30], yet no reports exist regarding its ability to synthesize biopolymers with industrial potential. This absence of data highlights an unexplored metabolic potential within A. luteoalbus, suggesting that novel strains could contribute to sustainable biopolymer development. The present study addresses this gap by isolating A. luteoalbus from the insect Ulomoides dermestoides , a new ecological niche that has not been previously explored for biopolymer production. The research aimed to evaluate its ability to produce biopolymers using agricultural residues such as orange peel, corncob, banana peel, prickly pear peel, and tejocote pulp as carbon sources, while optimizing culture conditions to improve material properties. The polymers obtained were characterized in terms of their chemical composition, mechanical and thermal behavior, and biodegradation potential, assessing their feasibility as sustainable alternatives that add value to organic waste while reducing reliance on synthetic plastics. 2. Methodology 2.1. Isolation and Identification of the Microorganism The fungus A. luteoalbus was isolated and purified from U. dermestoides using PDA and SDA media. The incubation temperature of 28 ± 2°C was chosen based on literature that indicates this range as optimal for filamentous fungi, providing favorable conditions for growth and metabolite production. Negative controls were included throughout to prevent environmental contamination. Initial identification was performed through macro- and microscopic morphology and further supported by molecular analysis of the 5.8S rRNA region. Although this fragment forms part of the ITS region commonly used as the fungal DNA barcode, its partial sequencing provides limited taxonomic resolution; therefore, the identification was interpreted with caution and consistent morphological characteristics of A. luteoalbus . The strain is preserved at the Industrial Microbiology Laboratory, FESC-UNAM. 2.2 Formulation of Culture Media for Biopolymer Production Six solid culture media were prepared using different carbon sources: orange peel, prickly pear peel, banana peel, tejocote pulp with peel, corncob, and sucrose. In all formulations, the organic substrate concentration was adjusted to 50 g/L, except for the tejocote pulp with peel, which was used at 30 g/L on a wet basis, and sucrose, which was used alone in medium 6 at 30 g/L. For all media, the following salts were added at constant concentrations: NaNO₃ 3 g/L, KH₂PO₄ 1 g/L, MgSO₄ 0.5 g/L, KCl 0.5 g/L, FeSO₄ 10 mg/L, and folic acid 0.166 g/L. In addition, agar 15 g/L was included in each formulation to obtain a solid medium. 2.3. Acid Hydrolysis of the Substrate The organic residues were washed, dried, ground, and weighed. They were then subjected to acid hydrolysis in an autoclave at 10 lb/in² for 20 min, adjusting the pH to 3.0 to release fermentable sugars. After hydrolysis, the mixture was allowed to stand, solids were removed by filtration, and the supernatant was retained for medium preparation. 2.4. Preparation of Culture Media The recovered supernatant was mixed with the salts and agar at the concentrations mentioned above. The final volume was adjusted to pH 6.0, clarified by heating, and sterilized in an autoclave at 15 lb/in² for 15 min. The sterilized medium was then cooled to 50 ± 2°C, and 35 ± 1 mL was poured into sterile Petri dishes (100 × 15 mm). The plates were left at room temperature until complete solidification before further use. 2.5. Biopolymer Production 2.5.1. Inoculation of the microorganism into the biopolymer production medium Once the culture plates were solidified at room temperature, they were inoculated by stab culture with A. luteoalbus and incubated at 28 ± 2°C under aerobic conditions for two and a half months. Growth and biopolymer formation were monitored weekly for a total of eleven weeks. Each substrate was prepared and inoculated in triplicate. 2.5.2. Recovery, purification of the biopolymer, and calculation of the percentage yield for each substrate studied Culture media containing the microorganism and the biopolymer were weighed weekly until the end of the eleven-week incubation period. At each sampling point, the biopolymers were recovered and sterilized in an autoclave at 15 lb/in² for 30 minutes. While still hot, the molten agar was removed, and the biopolymer was collected and subjected to reflux with distilled water for three hours to remove agar residues and other impurities. This purification process was repeated for each biopolymer obtained from the different substrates. Finally, the purified biopolymers were dried and weighed to determine yield. Results were expressed as the mean percentage of biopolymer ± the standard error of the mean over time, with each experiment conducted in triplicate (n = 3). 2.6. Biopolymer Characterization After purification, various analyses were performed to determine the properties of the biopolymers based on the carbon source used in their production. These analyses included scanning electron microscopy (SEM), melting point determination, combustion testing, solubility assessment, elemental analysis (CHNS), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The latter analyses were carried out at the Faculty of Chemistry, UNAM. 2.6.1. Scanning Electron Microscopy (SEM) To examine the microscopic morphology of the biopolymers, a JEOL JSM-6010LA scanning electron microscope was used under the following conditions: 15 kV accelerating voltage, 18 mm working distance, and 41 beam current, with images obtained at 10 µm scale and 2500× magnification. Samples collected at different growth stages were analyzed to compare the morphological characteristics of the biopolymers. 2.6.2. Physicochemical characterization Melting Point Determination The melting point was determined using a Fisher-Johns apparatus. A small piece of the biopolymer was placed between two round cover glasses and positioned on the heating stage. The equipment was switched on, the magnifying lens was aligned over the stage, and the rheostat knob was adjusted to increase the temperature. The sample and thermometer were observed simultaneously, and the melting temperature was recorded at the moment the solid melted. A maximum temperature of 250°C was recommended. The apparatus was then switched off and allowed to cool before reuse. Combustion Test Combustion behavior was evaluated using a Meker-Fisher burner. A small sample of the biopolymer was placed on a spatula and exposed to the flame. Odor, flame color, changes in the biopolymer color, and ash formation were recorded. Solubility Tests Solubility was assessed in glass tubes containing 1 mL of solvent. Fifty milligrams of biopolymer were added, and solubility was visually evaluated. The solvents tested were water, dimethyl sulfoxide (DMSO), 2-isopropanol, toluene, hexane, xylene, CCl₄, chloroform, ethyl acetate, and acetone. 2.6.3. Elemental Analysis (CHNS) Elemental analysis was performed in duplicate using a Perkin Elmer PE2400 Analyzer to determine the percentage of carbon, hydrogen, nitrogen, and sulfur. Approximately 2 mg of each sample was analyzed under the following parameters: Carrier/reference gas: Helium, Chromatographic column temperature: 82.2°C Detector: Thermal conductivity, Combustion reactor temperature: 975°C Reduction reactor temperature: 501°C, Analytical program: CHNS, Analysis time: 430 seconds, Calibration compound: Cystine (Perkin Elmer standard) 2.6.4 Thermal analysis Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) TGA was carried out using a Perkin Elmer TGA4000 equipped with Pyris software. The instrument was calibrated using Alumel, Perkalloy, and iron standards. Samples were weighed into 70 µL alumina pans and heated from 30 to 550°C at a 10°C/min heating rate under a nitrogen atmosphere. DSC was conducted using a Mettler Toledo DSC1 with STAR software version 14.0. The instrument was calibrated for temperature, heat flow, and total system accuracy. Samples were weighed into 40 µL aluminum pans, sealed with lids, perforated at the center, and heated from 25 to 550°C at a 10°C/min rate under nitrogen atmosphere. All analyses were performed with appropriate controls and statistical replication (n = 3) to ensure robust and reliable results. 3. Results 3.1. Identification of the microorganism by macroscopic and microscopic morphology The identification of the microorganism was based on both macroscopic and microscopic morphology. The macroscopic morphology of A. luteoalbus cultivated on different carbon sources at various incubation times is shown. Figures 1 illustrate colony characteristics, including growth pattern, color, and surface texture, which are used for the preliminary identification of the microorganism. Figure 2 shows the microscopic morphology of A. luteoalbus at 1000× magnification, observed with a Leica compound optical microscope (Model 1349521X) after staining with lactophenol cotton blue. The hyphae appear hyaline, thin, and septate, forming a branched mycelial network. Conidiophores are identifiable as erect structures that bear solitary or whorled phialides. From these phialides, unicellular conidia are produced, which are generally elliptical to sub-spherical, hyaline, and often exhibit a slight central curvature. In the examined fields, conidia were frequently arranged in compact aggregates at the apex of the phialides, while free conidia were also abundant, indicating an active stage of sporulation. These morphological features correspond with previous descriptions of A. luteoalbus [ 31 ]. 3.2 Identification of the microorganism by PCR The molecular analysis of the 5.8S rRNA region supported the identification of the fungal isolate as A. luteoalbus , showing 100% similarity with the reference sequence KT715723 available in the GenBank database. PCR amplicons were sequenced, and the resulting fragment was aligned against public databases using BLASTn. The alignment results are summarized in Table 1 . The single, high-confidence match obtained for A. luteoalbus indicated the absence of ambiguous alignments or cross-identification with related taxa. Although the sequenced region represents a conserved portion of the ITS locus and thus provides limited taxonomic resolution, the perfect match (together with the macro- and microscopic characteristics) confirms the reliability of the identification. Table 1 Molecular identification of Acrostalagmus luteoalbus based on partial 5.8S rRNA gene sequencing Aligned species Similarity (%) Accession number Country of origin Acrostalagmus luteoalbus 100 KT715723 Italy In summary, the sequencing and comparative analysis of the 5.8S fragment, supported by morphological evidence, provide consistent molecular confirmation of A. luteoalbus as the fungal isolate obtained from U. dermestoides . In Fig. 3 , the secretion of the extracellular biopolymer is observed directly under the stereomicroscope, confirming its localization outside the fungal cells. This extracellular production not only simplifies downstream processing but also suggests that the polymer is closely associated with the fungal network, which may contribute to the mechanical integrity of the material. The growth of A. luteoalbus is shown in Fig. 4 , where a thin extracellular polymer layer is clearly visible on the surface of the culture medium. This layer forms as the fungal mycelium develops, confirming that the biopolymer is secreted extracellularly rather than accumulated within the cells. Such extracellular localization offers significant advantages for downstream processing because it simplifies polymer recovery and avoids complex extraction procedures commonly required for intracellular polymers such as PHAs [ 32 ]. The washed biopolymers are shown in Fig. 5 , after being subjected to a three-hour reflux treatment to remove residual agar and impurities. Once dried, the biopolymer mass was recorded, and the yield percentage was calculated using the following Eq. 1 : Equation 1 \(\:\%\:Biopolymer=\:\frac{Biopolymer\:mass\:\left(g\right)}{Substrate\:mass\:\left(g\right)}x100\) The mean yields (Y p/s ± standard error) obtained for each substrate over the eleven-week period are summarized in Table 2 . The results indicate that maximum yields were reached at different incubation times depending on the substrate. A one-way ANOVA followed by Tukey's post hoc test was performed, considering differences significant at p ≤ 0.05. The data in Fig. 6 clearly demonstrate that all tested substrates supported biopolymer production, although yields varied greatly depending on both the substrate type and the incubation period. Among the substrates evaluated, “pulp with tejocote peel” achieved the highest yield (~ 17.10% ± 1.29) at week 9. Although a slight decline was observed by week 11 (~ 15.86% ± 1.58), this substrate consistently outperformed the others. These findings highlight week 9 as the optimal production point, suggesting that future scale-up or process optimization studies could focus on this time frame to maximize yields while avoiding unnecessary incubation periods that increase operational costs without improving productivity. Other substrates showed different yield profiles: Orange peel: increase until week 4 (8.24% ± 0.71), stabilization, and slight decrease by week 11 (7.87% ± 1.22), early linear fit R² ≈ 0.9473. Corncob: peak at week 7 (10.00% ± 0.45), stable yields around 9.25% ± 1.06, then decline at week 11 (7.89% ± 0.93), R² ≈ 0.9334. Prickly pear peel: increase until week 4 (9.32% ± 1.36), maximum at week 10 (10.15% ± 0.75), decrease by week 11 (8.68% ± 0.54), R² ≈ 0.8755. Banana peel: rise until week 9 (9.29% ± 0.63), followed by a decline to 7.30% ± 0.58 at week 11, R² ≈ 0.9402. Sucrose: nearly linear growth until week 10 (15.62% ± 0.77), slight reduction by week 11 (13.30% ± 1.37), R² ≈ 0.9451. Statistical analysis (ANOVA with Tukey’s post hoc test, p ≤ 0.05) revealed that prickly pear peel differed significantly from orange peel, while no significant differences were observed among the other substrates. Several factors likely account for the observed differences. The substrate composition is especially important: substrates containing both pulp and peel probably offer higher levels of simple sugars minerals, and moisture, promoting mycelial growth and biopolymer synthesis. Similarly, the availability of metabolizable carbon significantly influences polymer accumulation, with more accessible components promoting higher yields. These findings align with numerous studies that confirm agro-industrial residues such as fruit peels and crop processing byproducts are inexpensive, carbon-rich substrates for microbial biopolymer production [ 33 , 34 ]. However, yields are highly dependent on factors like microbial strain, substrate pretreatment, and operational parameters including pH, temperature, and aeration [ 35 , 36 ]. Beyond these, the physical characteristics of the substrates also play a critical role: more porous matrices enhance oxygen transfer and nutrient diffusion, whereas denser materials can restrict mass transfer and lower productivity. The stabilization or decline in yields after weeks 10–11 may be associated with nutrient depletion, accumulation of inhibitory metabolites, or unfavorable pH shifts, factors that have been shown to negatively affect PHA accumulation in mixed microbial cultures [ 37 ]. A particularly noteworthy finding is that all biopolymers obtained in this study were extracellular, regardless of the substrate. During fungal growth, a thin polymer layer was consistently observed on the surface of the culture medium [ 38 ]. This extracellular localization offers significant technical advantages over intracellular polymer production: it eliminates the need for cell disruption and solvent-intensive recovery, simplifying downstream processing and reducing costs [ 39 ]. These benefits are especially relevant when using low-cost agricultural residues, where minimizing operational costs is essential for ensuring economic feasibility [ 40 ]. When compared with the literature, most studies using fruit peels, bagasse, and other agricultural byproducts report biopolymer yields rarely exceeding 10–15% without prior substrate pretreatment or intensive process optimization [ 41 ]. As highlighted in recent studies, the efficiency of microbial bioproduct generation from agroindustrial waste depends strongly on the substrate composition and fermentation method, particularly when using low-cost materials such as fruit residues. Therefore, the ~ 17% yield obtained with the tejocote peel substrate in this study is particularly remarkable, as it exceeds most previously reported values and highlights week 9 as a key time point for process optimization. In summary, the data suggest that: Substrate selection critically affects biopolymer yields, extracellular production by A. luteoalbus provides technical and economic advantages over intracellular systems, and further optimization of pH, substrate concentration, aeration, and agitation could potentially enhance productivity and product quality even more. 3.3 Scanning Electron Microscopy (SEM) Figure 7 displays scanning electron micrographs (SEM) taken at 500× magnification, using an accelerating voltage of 15 kV and a working distance of 18 mm, with a JEOL JSM-6010LA microscope. The analysis was performed to assess the impact of substrate composition and fermentation time on the biopolymer's microstructure. The observed morphological features in these SEM images, including variations in surface arrangement, fiber formation, and density, are further summarized in Table 2 , which correlates these microstructural changes with mechanical properties and yield outcomes for each substrate. The comparative analysis of how substrate type and fermentation time influence the microstructural development, mechanical properties, and yield of the resulting biopolymers is shown in Table 2 . Substrates rich in simple sugars, such as sucrose and tejocote peel pulp, promote the formation of uniform and durable films, while lignocellulosic residues like corncob and banana peel produce amorphous or brittle polymers with lower mechanical performance. These results confirm that substrate composition directly affects both the kinetics of polymer formation and the structural integrity of the material [ 42 , 43 ]. Table 2 Comparative Summary relationship between substrate type, fermentation time, microstructural evolution, mechanical properties, and polymer yields Substrate Initial Week Initial Microstructure Final Week Final Microstructure Mechanical Properties Yield Orange peel 7 weeks Protuberances, partial transformation 10 weeks Continuous, uniform film High resistance Medium Corncob 5 weeks Amorphous, poorly organized structure 10 weeks Similar morphology, no major changes Low resistance Low Prickly pear peel 5 weeks Compact film 10 weeks Brittle, fragmented structure Reduced integrity over time Medium Tejocote peel pulp 5 weeks Fibrous, incomplete transformation 10 weeks Continuous, uniform film High resistance High Banana peel 7 weeks Compact film 10 weeks Fibrous, brittle structure Reduced integrity over time Medium Sucrose 1 week Scaly, compact structure 10 weeks Thin, resistant, flexible film High resistance & flexibility High The comparative analysis in Table 2 shows that substrate type and fermentation time are crucial for the microstructural development, mechanical properties, and yield of biopolymers. Consistent with previous studies [ 44 , 45 ], substrates high in simple sugars, such as sucrose and tejocote peel pulp, support the quick formation of uniform, continuous, and mechanically strong films. In both cases, media containing readily metabolizable carbon sources led to faster polymer synthesis and better structural integrity of the final product, highlighting the significant impact of substrate composition on microbial biopolymer performance. This observation aligns with earlier reports indicating that the carbon source not only dictates microbial metabolic efficiency but also modulates polymer crystallinity and porosity, thereby determining its mechanical performance and potential functional applications [ 46 , 47 ]. Compared to thermally compressed synthetic polymers like High-Density Polyethylene (HDPE), which show a compact, uniform, and low-porosity structure associated with high mechanical strength and low degradability [ 48 ], polyhydroxyalkanoates (PHAs) display a more amorphous, porous, and irregular microstructure resulting from direct microbial biosynthesis without additional thermal processing [ 49 ]. This open structure, marked by interconnected cavities and uneven surfaces, is commonly reported in PHAs used in medical and agricultural applications [ 50 ]. Notably, the natural porosity of PHAs is linked to increased water absorption and molecular diffusion, properties that support controlled-release mechanisms in drug delivery and interactions with biological tissues [ 50 ]. Furthermore, the microbial origin and biodegradable composition of PHAs enhance their environmental and physiological degradation compared to denser or more crystalline biopolymers. These combined attributes—biodegradability, structural compatibility, and tunable porosity—have driven their development in applications ranging from regenerative medicine and smart packaging to sustainable agricultural systems [ 51 , 52 ]. 3.4 Physicochemical Properties The results summarized in Table 3 demonstrate that all biopolymers obtained from different carbon sources exhibited similar physicochemical properties, including a dark brown, opaque appearance, brittle texture, and resistance to scratching. The melting point above 250°C indicates that the materials possess high thermal stability, a desirable feature for packaging and biomedical applications requiring dimensional stability at elevated temperatures. The combustion behavior, marked by a red flame, sweet odor during burning, and carbonization when removed from the flame, indicates a carbon-rich structure with high thermal resistance, aligning with findings for other fungal-derived biopolymers [ 53 ]. Moreover, the complete insolubility of all samples in both polar and non-polar solvents, including water, dimethyl sulfoxide, chloroform, and acetone, suggests a highly cross-linked or partially crystalline macromolecular network, like chitin and fungal melanins, which show comparable insolubility patterns [ 54 ]. Although this insolubility may limit some processing methods, it could provide benefits for water-resistant or barrier applications where solvent stability is advantageous. Table 3 Experimental results of physicochemical tests performed using different carbon sources Test performed Result Initial observations Opaque, plastic-like, brittle, resistant to scratching, dark brown color. Melting point > 250°C Combustion In flame: red color, sweet odor, remains ignited. Outside flame: white color, pungent odor, with presence of ash. Solubility Insoluble in water, dimethyl sulfoxide (DMSO), 2-isopropanol, toluene, hexane, xylene, carbon tetrachloride (CCl₄), chloroform, ethyl acetate, acetone. 3.5 Elemental Analysis (CHNS) The elemental composition of the biopolymer obtained from the sucrose medium after eleven weeks is shown in Table 4 . The polymer contained 44.5% carbon, 7.425% hydrogen, 1.80% nitrogen, and 0.605% sulfur, with the remainder likely consisting of oxygen and trace inorganic elements not measured by this method. The low nitrogen and sulfur content indicates a mainly carbohydrate- or polyphenolic-based composition rather than a protein-rich structure. The carbon fraction aligns with values reported for microbial polysaccharides and fungal cell wall polymers, supporting the hypothesis that the material comprises complex polysaccharides with both aromatic and aliphatic domains. Such compositions are typical for fungal exopolysaccharides, which frequently demonstrate thermal resilience and low solubility due to extensive hydrogen bonding and structural heterogeneity. Table 4 Elemental analysis results obtained with the Perkin Elmer 2400 Elemental Analyzer Sample amount analyzed (mg) % Carbon % Hydrogen % Nitrogen % Sulfur 2.606 44.41 7.52 1.82 0.57 2.317 44.59 7.33 1.78 0.64 Mean ± SD (n = 2) 44.50 ± 0.13 7.43 ± 0.13 1.80 ± 0.03 0.61 ± 0.05 3.6 Thermal Analysis (TGA and DSC) The TGA thermogram (Table 5 ; Fig. 8 ) reveals three main stages of weight loss. The initial decrease below 120°C, corresponding to approximately 19.14% mass loss, reflects the evaporation of adsorbed and interlaminar water molecules, indicating a high moisture retention capacity within the polymeric network. The subsequent degradation phase between 250°C and 400°C shows three defined peaks at 286.20°C, 316.84°C, and 390.46°C, suggesting a stepwise breakdown of polysaccharidic components and associated organic moieties. This multistage behavior denotes structural heterogeneity and the coexistence of both thermolabile and thermoresistant domains within the matrix. Beyond 450°C, the mass loss rate decreases markedly, leaving a stable carbonaceous residue that points to the presence of aromatic or cross-linked fractions conferring enhanced thermal resilience to the biopolymer. Table 5 Thermogravimetric analysis (TGA) results obtained with the Perkin Elmer TGA4000 Parameter Result Initial sample mass (mg) 3.374 Maximum temperature – 1st transition (°C) 35.710 Mass loss – 1st transition (%) 6.211 Maximum temperature – 2nd transition (°C) 54.770 Mass loss – 2nd transition (%) 12.929 Maximum temperature – 3rd transition (°C) 286.200 Mass loss – 3rd transition (%) 8.376 Maximum temperature – 4th transition (°C) 316.840 Mass loss – 4th transition (%) 28.017 Maximum temperature – 5th transition (°C) 390.460 Mass loss – 5th transition (%) 17.241 The DSC thermogram (Table 6 ; Fig. 9 ) further corroborates these findings. The glass transition temperature (Tg) at 197.52°C marks the transition from a rigid, brittle state to a soft, flexible material. The softening range (200–250°C) preceded major degradation, indicating a narrow processing window for applications requiring thermal shaping or sterilization Beyond 250°C, progressive endothermic events were observed at 416°C and 546°C, associated with structural decomposition rather than melting transitions. The absence of melting peaks suggests that the polymer either does not melt under the tested conditions (30–550°C) or transitions directly from the solid to the degradation phase. Table 6 Differential Scanning Calorimetry (DSC) results obtained with the Mettler Toledo DSC1 1st transition (°C) 2nd transition (°C) 3rd transition (°C) 4th transition (°C) 5th transition (°C) 6th transition (°C) Onset of melting = 31.47°C Peak melting temperature = 63.20°C Two-components temperature = 123.82°C Enthalpy = -260.07 J/g 84.52 197.52 274.10 416.38 546.67 The high thermal stability, as demonstrated by onset degradation temperatures above 250°C and no melting transitions, indicates that this polymer could be suitable for biodegradable packaging materials, thermal insulation components, or biomedical membranes requiring dimensional stability under heat exposure. The sequential decomposition peaks suggest a heterogeneous macromolecular architecture, where different polysaccharide fractions or cross-linked domains degrade in stages, a phenomenon widely reported for microbial exopolysaccharides and lignocellulosic biopolymers [ 55 ]. Furthermore, the insolubility across all tested solvents combined with thermal resilience implies a heavily hydrogen-bonded or partially crystalline structure, like fungal melanins or chitin derivatives, which exhibit remarkable chemical and thermal resistance and often require specialized processing routes for functionalization [ 56 ]. The effect of carbon source on polymer properties merits further investigation. Although sucrose produced the material analyzed here, fruit residues such as tecojote peel provided higher biopolymer yields in production trials; whether this also affects thermal or chemical properties should be explored in future studies. Finally, despite the outstanding properties of the biopolymer (particularly its thermal stability and chemical resistance) it is essential to acknowledge several limitations that currently restrict its broader applicability. The material exhibited a brittle texture, indicating low flexibility and constraining its use in applications requiring elasticity or tensile strength, such as flexible packaging or biodegradable films. Additionally, the dark coloration observed may represent both an aesthetic and technological barrier for its incorporation into food packaging or biomedical applications where transparency and visual appearance are critical. Nevertheless, biopolymer bleaching strategies could potentially overcome this limitation. Another important constraint is its insolubility in all tested solvents, a feature that, while conferring stability under extreme conditions, significantly limits processing through conventional molding or extrusion techniques. Taken together, these findings suggest that, in its native form, the biopolymer is more suitable for specialized applications such as protective coatings, chemically resistant membranes, or barrier materials rather than as a direct substitute for mass-consumption plastics like polyethylene or polypropylene. Further research is required to address these limitations through chemical modifications, blending strategies, or advanced processing methods to enhance the material’s flexibility, transparency, and processability, thereby expanding its potential industrial applications. Conclusions The results of this study demonstrated that A. luteoalbus is capable of producing an extracellular biopolymer using low-cost carbon sources, including agro-industrial residues, representing a sustainable strategy for the valorization of organic waste. The highest yield (~ 17.10% ± 1.29 at week 9 with pulp and tejocote peel) confirmed that substrate composition and incubation time are critical factors influencing the productivity of the process. Physicochemical, elemental, and thermal analyses showed that the obtained material exhibits high thermal stability, insolubility in common solvents, and resistance to degradation at temperatures above 250°C. These properties, together with its extracellular nature that simplifies recovery, position the biopolymer as a potential candidate for applications in biodegradable packaging, protective coatings, and biomedical materials, where chemical and thermal resistance are essential. Declarations Perspectives Future work should focus on optimizing culture parameters such as pH, aeration, substrate concentration, and agitation conditions to further enhance productivity and evaluate process scalability in larger bioreactors. Additionally, it will be essential to conduct in-depth structural characterizationusing advanced spectroscopic (FTIR, NMR) and rheological techniques, as well as to assess mechanical properties , biodegradability , and performance under real-use conditions . Such studies will help establish the technical, economic, and environmental feasibility of producing this biopolymer at an industrial scale, contributing to reducing the use of petroleum-derived plastics and promoting the development of a sustainable circular economy. Authors’ Contributions/Notes/Thanks/Other declarations All authors contributed equally to writing the article. Additionally, all authors have reviewed and approved the final version for publication. Notes/Thanks/Other declarations All authors contributed equally to writing the article. Additionally, all authors have reviewed and approved the final version for publication. Funding No funding was received for this study. 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05:52:40","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165052,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/fdcfd25fb54931c8154dd787.html"},{"id":94059483,"identity":"c5d94114-efd7-4dff-b4da-5ce5d15552a8","added_by":"auto","created_at":"2025-10-22 06:08:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2102736,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic growth of \u003cem\u003eA. luteoalbus\u003c/em\u003e on different carbon sources: orange peel, corncob, prickly pear peel, pulp with tejocote peel, banana peel, and sucrose. at various incubation times\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/7ba9a1072d454b9672f98b46.png"},{"id":94060167,"identity":"adc8ab06-473f-43c4-8b44-af34c8054903","added_by":"auto","created_at":"2025-10-22 06:16:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2658946,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic morphology of \u003cem\u003eA. luteoalbus\u003c/em\u003e using lactophenol cotton blue staining. (a) Hyaline, septate hyphae with initial clusters of conidia. (b) Branched hyphae bearing phialides with aggregated conidia in a whorled arrangement. (c) Erect phialides producing compact groups of elliptical conidia. (d) Numerous free conidia were released into the medium, consistent with advanced sporulation\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/33c611c358285ccae67bd293.png"},{"id":94058721,"identity":"789a54dc-91ce-4b2e-996f-1f035f7c3b95","added_by":"auto","created_at":"2025-10-22 05:52:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":959130,"visible":true,"origin":"","legend":"\u003cp\u003eSecretion of extracellular biopolymer by \u003cem\u003eA. luteoalbus\u003c/em\u003eobserved under a stereomicroscope at 4× magnification (BOECO Germany, model BOE3200.001). The polymeric material appears as bright yellowish aggregates secreted outside the fungal cells\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/f58c43a53816a9cc838ac742.png"},{"id":94058725,"identity":"66294c3a-9244-4b87-9cd3-75d79b5eab07","added_by":"auto","created_at":"2025-10-22 05:52:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45942,"visible":true,"origin":"","legend":"\u003cp\u003eFormation of a thin extracellular biopolymer layer on the surface of the culture medium during \u003cem\u003eA. luteoalbus\u003c/em\u003e growth. The extracellular localization provides operational advantages by making polymer recovery easier\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/84851ea15f9264b3ad1051d0.jpg"},{"id":94059484,"identity":"c975fa68-9fbc-4a4c-8dca-cd7d643732fa","added_by":"auto","created_at":"2025-10-22 06:08:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3061218,"visible":true,"origin":"","legend":"\u003cp\u003eWashed biopolymers obtained after three hours of reflux treatment to remove residual agar prior to drying and yield determination\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/5549796adfd4fe3184732a9f.png"},{"id":94059025,"identity":"053a7622-e93d-47dd-aff9-1d8de3f32bf7","added_by":"auto","created_at":"2025-10-22 06:00:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":851206,"visible":true,"origin":"","legend":"\u003cp\u003eAverage yield of biopolymer production (Yp/s, % ± SE) using different carbon sources over 11 weeks of fermentation. Error bars represent the standard error of the mean (n=3). Substrates tested: orange peel, corncob, prickly pear peel, pulp with tejocote peel, banana peel, and sucrose\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/a414bd401298f64e98a313a0.jpg"},{"id":94058751,"identity":"79e84939-70a1-4e01-b5f1-f036e5773e45","added_by":"auto","created_at":"2025-10-22 05:52:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7127011,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) micrographs of biopolymers obtained from different substrates: (a, b) sucrose; (c, d) orange peel; (e, f) corncob; (g, h) banana peel; (i, j) prickly pear peel; (k, l) tejocote pulp with peel. All images were acquired at 15 kV, with a working distance of 17 mm, magnifications ranging from ×500 to ×2000, and a scale bar of 50 μm. Morphological variations in surface structure and fiber formation can be observed depending on the substrate used\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/9192b169fcad7784b7edbeec.png"},{"id":94060427,"identity":"f2f6d313-8478-4b4c-b553-c68efe848683","added_by":"auto","created_at":"2025-10-22 06:24:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":150772,"visible":true,"origin":"","legend":"\u003cp\u003eThermogram obtained from thermogravimetric analysis (TGA). Mass percentage vs. temperature.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/d40aa5dcb50cd27c39a72bc2.png"},{"id":94059022,"identity":"9cbbf03e-0a27-4b2e-92ce-728cd611825d","added_by":"auto","created_at":"2025-10-22 06:00:40","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":85008,"visible":true,"origin":"","legend":"\u003cp\u003eThermogram obtained from Differential Scanning Calorimetry (DSC) analysis. Heat flow vs temperature\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/9b20c4ac55749377f655c6f9.jpg"},{"id":94290487,"identity":"8d4b698b-647e-4ab0-9324-e9bc1f89a988","added_by":"auto","created_at":"2025-10-27 11:19:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20825979,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/570b6b50-55cb-4ef0-91d0-6d2ffce4808e.pdf"},{"id":94058727,"identity":"62efa3f6-a99c-46e2-9f29-6c84d64e4e1b","added_by":"auto","created_at":"2025-10-22 05:52:40","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9251342,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7901410/v1/b281dfd8cd7878d80b486853.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eExtracellular Biopolymer Production by \u003cem\u003eAcrostalagmus luteoalbus\u003c/em\u003efrom Agro-Industrial Wastes: Toward Sustainable Material Development\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe synthesis and widespread use of synthetic polymers revolutionized modern life in the mid-20th century. Due to their versatility, low cost, lightness, and durability, plastics became ubiquitous in packaging, textiles, construction, automotive, and consumer goods [1]. However, these same properties also cause severe environmental problems, since plastics persist for decades or centuries in soils, oceans, and the atmosphere, accumulating as hard-to-manage waste. Global plastic production has increased dramatically from around 2 million tons in 1950 to over 450 million tons in 2023 [2,3]. Annual plastic waste generation now exceeds 350 million tons worldwide [3,4]. Once in the environment, plastics fragment into microplastics that enter food chains and affect ecosystems and potentially human health [5]. Inadequate disposal of plastic waste leads to soil degradation, wildlife mortality through accidental ingestion, and the release of toxic emissions (especially dioxins and furans) during uncontrolled incineration [6,7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlastics exhibit extreme persistence, often lasting for centuries in the environment [8,9], highlighting the urgent need for sustainable alternatives. Plastic pollution has become a global crisis, with an estimated 19 to 23 million tons of plastic waste leaking into aquatic ecosystems each year, polluting rivers, lakes, and oceans, and posing severe threats to biodiversity, coastal economies, and human health [10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this context, these materials have gained attention as sustainable alternatives to conventional plastics [11]. They consist of macromolecules derived partially or entirely from renewable biological sources (such as plants, algae, and microorganisms) and typically exhibit biodegradability and biocompatibility [12,13].\u0026nbsp;Bioplastics can be engineered to integrate into circular systems such as reuse, composting, anaerobic digestion, and organic recycling, thereby reducing reliance on fossil resources and helping to close carbon cycles [14,15].\u0026nbsp;Biodegradation generally involves the breakdown of polymer chains by microorganisms into carbon dioxide, water, and biomass under aerobic conditions, or methane under anaerobic conditions. These biological processes are often complemented by abiotic mechanisms such as hydrolytic, thermal, and photo-oxidative degradation, depending on the polymer’s chemical structure and environmental factors [16,17].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiopolymers can be broadly classified into four categories: (i) natural renewable polymers such as starch, cellulose, and chitin; (ii) microbial polymers, including polyhydroxyalkanoates (PHAs); (iii) chemically synthesized polymers derived from renewable biomonomers such as polylactic acid (PLA); and (iv) biodegradable petrochemical-based polymers, such as polycaprolactone (PCL) [18,19]. Within these,\u0026nbsp;polyhydroxyalkanoates\u0026nbsp;(PHAs) such as polyhydroxybutyrate (PHB) have been extensively studied as biodegradable thermoplastics, with growing research directed toward low-cost feedstocks like lignocellulosic biomass and agro-industrial residues [20–22]. Despite these advances, industrial-scale PHA production remains limited due to high production costs, low yields, and downstream processing challenges [20,23]. In contrast, filamentous fungi remain underexplored as PHA producers, even though they exhibit strong metabolic adaptability and can utilize complex carbon sources such as agro-wastes and food residues [24].\u003c/p\u003e\n\u003cp\u003eGiven these limitations, exploring alternative microbial taxa with unexplored biosynthetic capabilities is crucial for expanding the range of biopolymer-producing organisms.Recent studies have explored the valorization of agro-industrial residues for microbial biopolymer production. Selvam et al. [25] reviewed the potential of agricultural residues such as sugarcane bagasse, rice husks, banana peels, and spent mushroom substrate for microbial fermentations aimed at biopolymer synthesis. Similarly, Jørgensen et al. [26] analyzed enzymatic approaches for lignocellulose conversion into fermentable sugars, while Barua et al. [27] discussed advances in the sustainable transformation of lignocellulosic wastes into bioplastic precursors. These valorization strategies not only reduce raw material costs but also mitigate the environmental burden associated with waste disposal, reinforcing the transition toward sustainable bioeconomies.\u003c/p\u003e\n\u003cp\u003eMoreover, bioplastics have broad applications, ranging from food packaging, disposable utensils, and textiles to biomedical devices, sutures, drug-delivery systems, and agricultural films [28]. Despite these advances, no microbial biopolymer has yet matched the mechanical or thermal performance of polyethylene, polypropylene, or PET, and challenges remain in improving durability, stability, and lowering costs [28].\u003c/p\u003e\n\u003cp\u003eThe genus \u003cem\u003eAcrostalagmus\u003c/em\u003e is recognized for its metabolic versatility and production of diverse metabolites, including antimicrobials and toxins [29]. \u003cem\u003eAcrostalagmus luteoalbus\u003c/em\u003e has been identified in damp environments and building materials [30], yet no reports exist regarding its ability to synthesize biopolymers with industrial potential. This absence of data highlights an unexplored metabolic potential within \u003cem\u003eA.\u003c/em\u003e\u003cem\u003e\u0026nbsp;luteoalbus,\u003c/em\u003e suggesting that novel strains could contribute to sustainable biopolymer development.\u003c/p\u003e\n\u003cp\u003eThe present study addresses this gap by isolating \u003cem\u003eA. luteoalbus\u003c/em\u003e from the insect \u003cem\u003eUlomoides dermestoides\u003c/em\u003e, a new ecological niche that has not been previously explored for biopolymer production. The research aimed to evaluate its ability to produce biopolymers using agricultural residues such as orange peel, corncob, banana peel, prickly pear peel, and tejocote pulp as carbon sources, while optimizing culture conditions to improve material properties. The polymers obtained were characterized in terms of their chemical composition, mechanical and thermal behavior, and biodegradation potential, assessing their feasibility as sustainable alternatives that add value to organic waste while reducing reliance on synthetic plastics.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Isolation and Identification of the Microorganism\u003c/h2\u003e\u003cp\u003eThe fungus \u003cem\u003eA. luteoalbus\u003c/em\u003e was isolated and purified from \u003cem\u003eU. dermestoides\u003c/em\u003e using PDA and SDA media. The incubation temperature of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C was chosen based on literature that indicates this range as optimal for filamentous fungi, providing favorable conditions for growth and metabolite production. Negative controls were included throughout to prevent environmental contamination. Initial identification was performed through macro- and microscopic morphology and further supported by molecular analysis of the 5.8S rRNA region. Although this fragment forms part of the ITS region commonly used as the fungal DNA barcode, its partial sequencing provides limited taxonomic resolution; therefore, the identification was interpreted with caution and consistent morphological characteristics of \u003cem\u003eA. luteoalbus\u003c/em\u003e. The strain is preserved at the Industrial Microbiology Laboratory, FESC-UNAM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Formulation of Culture Media for Biopolymer Production\u003c/h2\u003e\u003cp\u003eSix solid culture media were prepared using different carbon sources: orange peel, prickly pear peel, banana peel, tejocote pulp with peel, corncob, and sucrose. In all formulations, the organic substrate concentration was adjusted to 50 g/L, except for the tejocote pulp with peel, which was used at 30 g/L on a wet basis, and sucrose, which was used alone in medium 6 at 30 g/L.\u003c/p\u003e\u003cp\u003eFor all media, the following salts were added at constant concentrations: NaNO₃ 3 g/L, KH₂PO₄ 1 g/L, MgSO₄ 0.5 g/L, KCl 0.5 g/L, FeSO₄ 10 mg/L, and folic acid 0.166 g/L. In addition, agar 15 g/L was included in each formulation to obtain a solid medium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Acid Hydrolysis of the Substrate\u003c/h2\u003e\u003cp\u003eThe organic residues were washed, dried, ground, and weighed. They were then subjected to acid hydrolysis in an autoclave at 10 lb/in\u0026sup2; for 20 min, adjusting the pH to 3.0 to release fermentable sugars. After hydrolysis, the mixture was allowed to stand, solids were removed by filtration, and the supernatant was retained for medium preparation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of Culture Media\u003c/h2\u003e\u003cp\u003eThe recovered supernatant was mixed with the salts and agar at the concentrations mentioned above. The final volume was adjusted to pH 6.0, clarified by heating, and sterilized in an autoclave at 15 lb/in\u0026sup2; for 15 min. The sterilized medium was then cooled to 50\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mL was poured into sterile Petri dishes (100 \u0026times; 15 mm). The plates were left at room temperature until complete solidification before further use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Biopolymer Production\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Inoculation of the microorganism into the biopolymer production medium\u003c/h2\u003e\u003cp\u003eOnce the culture plates were solidified at room temperature, they were inoculated by stab culture with \u003cem\u003eA. luteoalbus\u003c/em\u003e and incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under aerobic conditions for two and a half months. Growth and biopolymer formation were monitored weekly for a total of eleven weeks. Each substrate was prepared and inoculated in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Recovery, purification of the biopolymer, and calculation of the percentage yield for each substrate studied\u003c/h2\u003e\u003cp\u003eCulture media containing the microorganism and the biopolymer were weighed weekly until the end of the eleven-week incubation period. At each sampling point, the biopolymers were recovered and sterilized in an autoclave at 15 lb/in\u0026sup2; for 30 minutes.\u003c/p\u003e\u003cp\u003eWhile still hot, the molten agar was removed, and the biopolymer was collected and subjected to reflux with distilled water for three hours to remove agar residues and other impurities. This purification process was repeated for each biopolymer obtained from the different substrates.\u003c/p\u003e\u003cp\u003eFinally, the purified biopolymers were dried and weighed to determine yield. Results were expressed as the mean percentage of biopolymer\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard error of the mean over time, with each experiment conducted in triplicate (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Biopolymer Characterization\u003c/h2\u003e\u003cp\u003eAfter purification, various analyses were performed to determine the properties of the biopolymers based on the carbon source used in their production. These analyses included scanning electron microscopy (SEM), melting point determination, combustion testing, solubility assessment, elemental analysis (CHNS), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The latter analyses were carried out at the Faculty of Chemistry, UNAM.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1. Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eTo examine the microscopic morphology of the biopolymers, a JEOL JSM-6010LA scanning electron microscope was used under the following conditions: 15 kV accelerating voltage, 18 mm working distance, and 41 beam current, with images obtained at 10 \u0026micro;m scale and 2500\u0026times; magnification. Samples collected at different growth stages were analyzed to compare the morphological characteristics of the biopolymers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2. Physicochemical characterization\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eMelting Point Determination\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe melting point was determined using a Fisher-Johns apparatus. A small piece of the biopolymer was placed between two round cover glasses and positioned on the heating stage. The equipment was switched on, the magnifying lens was aligned over the stage, and the rheostat knob was adjusted to increase the temperature. The sample and thermometer were observed simultaneously, and the melting temperature was recorded at the moment the solid melted. A maximum temperature of 250\u0026deg;C was recommended. The apparatus was then switched off and allowed to cool before reuse.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eCombustion Test\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eCombustion behavior was evaluated using a Meker-Fisher burner. A small sample of the biopolymer was placed on a spatula and exposed to the flame. Odor, flame color, changes in the biopolymer color, and ash formation were recorded.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSolubility Tests\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eSolubility was assessed in glass tubes containing 1 mL of solvent. Fifty milligrams of biopolymer were added, and solubility was visually evaluated. The solvents tested were water, dimethyl sulfoxide (DMSO), 2-isopropanol, toluene, hexane, xylene, CCl₄, chloroform, ethyl acetate, and acetone.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3. Elemental Analysis (CHNS)\u003c/h2\u003e\u003cp\u003eElemental analysis was performed in duplicate using a Perkin Elmer PE2400 Analyzer to determine the percentage of carbon, hydrogen, nitrogen, and sulfur. Approximately \u003cb\u003e2 mg\u003c/b\u003e of each sample was analyzed under the following parameters:\u003c/p\u003e\u003cp\u003eCarrier/reference gas: Helium, Chromatographic column temperature: 82.2\u0026deg;C\u003c/p\u003e\u003cp\u003eDetector: Thermal conductivity, Combustion reactor temperature: 975\u0026deg;C\u003c/p\u003e\u003cp\u003eReduction reactor temperature: 501\u0026deg;C, Analytical program: CHNS, Analysis time: 430 seconds, Calibration compound: Cystine (Perkin Elmer standard)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4 Thermal analysis\u003c/h2\u003e\u003cp\u003e\u003cb\u003eThermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTGA was carried out using \u003cb\u003ea\u003c/b\u003e Perkin Elmer TGA4000 equipped with Pyris software. The instrument was calibrated using Alumel, Perkalloy, and iron standards. Samples were weighed into 70 \u0026micro;L alumina pans and heated from 30 to 550\u0026deg;C at a 10\u0026deg;C/min heating rate under a nitrogen atmosphere.\u003c/p\u003e\u003cp\u003eDSC was conducted using \u003cb\u003ea\u003c/b\u003e Mettler Toledo DSC1 with STAR software version 14.0. The instrument was calibrated for temperature, heat flow, and total system accuracy. Samples were weighed into 40 \u0026micro;L aluminum pans, sealed with lids, perforated at the center, and heated from 25 to 550\u0026deg;C at a 10\u0026deg;C/min rate under nitrogen atmosphere. All analyses were performed with appropriate controls and statistical replication (n\u0026thinsp;=\u0026thinsp;3) to ensure robust and reliable results.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Identification of the microorganism by macroscopic and microscopic morphology\u003c/h2\u003e\u003cp\u003eThe identification of the microorganism was based on both macroscopic and microscopic morphology. The macroscopic morphology of \u003cem\u003eA. luteoalbus\u003c/em\u003e cultivated on different carbon sources at various incubation times is shown. Figures\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrate colony characteristics, including growth pattern, color, and surface texture, which are used for the preliminary identification of the microorganism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the microscopic morphology of \u003cem\u003eA. luteoalbus\u003c/em\u003e at 1000\u0026times; magnification, observed with a Leica compound optical microscope (Model 1349521X) after staining with lactophenol cotton blue. The hyphae appear hyaline, thin, and septate, forming a branched mycelial network. Conidiophores are identifiable as erect structures that bear solitary or whorled phialides. From these phialides, unicellular conidia are produced, which are generally elliptical to sub-spherical, hyaline, and often exhibit a slight central curvature. In the examined fields, conidia were frequently arranged in compact aggregates at the apex of the phialides, while free conidia were also abundant, indicating an active stage of sporulation. These morphological features correspond with previous descriptions of \u003cem\u003eA. luteoalbus\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Identification of the microorganism by PCR\u003c/h2\u003e\u003cp\u003eThe molecular analysis of the 5.8S rRNA region supported the identification of the fungal isolate as \u003cem\u003eA. luteoalbus\u003c/em\u003e, showing 100% similarity with the reference sequence KT715723 available in the GenBank database. PCR amplicons were sequenced, and the resulting fragment was aligned against public databases using BLASTn. The alignment results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The single, high-confidence match obtained for \u003cem\u003eA. luteoalbus\u003c/em\u003e indicated the absence of ambiguous alignments or cross-identification with related taxa. Although the sequenced region represents a conserved portion of the ITS locus and thus provides limited taxonomic resolution, the perfect match (together with the macro- and microscopic characteristics) confirms the reliability of the identification.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular identification of Acrostalagmus luteoalbus based on partial 5.8S rRNA gene sequencing\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAligned species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimilarity\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAccession number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCountry of\u003c/p\u003e\u003cp\u003eorigin\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAcrostalagmus luteoalbus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKT715723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eItaly\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn summary, the sequencing and comparative analysis of the 5.8S fragment, supported by morphological evidence, provide consistent molecular confirmation of \u003cem\u003eA. luteoalbus\u003c/em\u003e as the fungal isolate obtained from \u003cem\u003eU. dermestoides\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the secretion of the extracellular biopolymer is observed directly under the stereomicroscope, confirming its localization outside the fungal cells. This extracellular production not only simplifies downstream processing but also suggests that the polymer is closely associated with the fungal network, which may contribute to the mechanical integrity of the material.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe growth of \u003cem\u003eA. luteoalbus\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, where a thin extracellular polymer layer is clearly visible on the surface of the culture medium. This layer forms as the fungal mycelium develops, confirming that the biopolymer is secreted extracellularly rather than accumulated within the cells. Such extracellular localization offers significant advantages for downstream processing because it simplifies polymer recovery and avoids complex extraction procedures commonly required for intracellular polymers such as PHAs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe washed biopolymers are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, after being subjected to a three-hour reflux treatment to remove residual agar and impurities. Once dried, the biopolymer mass was recorded, and the yield percentage was calculated using the following \u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e:\u003c/p\u003e\u003cp\u003e\u003cb\u003eEquation 1\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\%\\:Biopolymer=\\:\\frac{Biopolymer\\:mass\\:\\left(g\\right)}{Substrate\\:mass\\:\\left(g\\right)}x100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe mean yields (Y\u003csub\u003ep/s\u003c/sub\u003e \u0026plusmn; standard error) obtained for each substrate over the eleven-week period are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results indicate that maximum yields were reached at different incubation times depending on the substrate. A one-way ANOVA followed by Tukey's post hoc test was performed, considering differences significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe data in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e clearly demonstrate that all tested substrates supported biopolymer production, although yields varied greatly depending on both the substrate type and the incubation period. Among the substrates evaluated, \u0026ldquo;pulp with tejocote peel\u0026rdquo; achieved the highest yield (~\u0026thinsp;17.10% \u0026plusmn; 1.29) at week 9. Although a slight decline was observed by week 11 (~\u0026thinsp;15.86% \u0026plusmn; 1.58), this substrate consistently outperformed the others. These findings highlight week 9 as the optimal production point, suggesting that future scale-up or process optimization studies could focus on this time frame to maximize yields while avoiding unnecessary incubation periods that increase operational costs without improving productivity.\u003c/p\u003e\u003cp\u003eOther substrates showed different yield profiles:\u003c/p\u003e\u003cp\u003eOrange peel: increase until week 4 (8.24% \u0026plusmn; 0.71), stabilization, and slight decrease by week 11 (7.87% \u0026plusmn; 1.22), early linear fit R\u0026sup2; \u0026asymp; 0.9473.\u003c/p\u003e\u003cp\u003eCorncob: peak at week 7 (10.00% \u0026plusmn; 0.45), stable yields around 9.25% \u0026plusmn; 1.06, then decline at week 11 (7.89% \u0026plusmn; 0.93), R\u0026sup2; \u0026asymp; 0.9334.\u003c/p\u003e\u003cp\u003ePrickly pear peel: increase until week 4 (9.32% \u0026plusmn; 1.36), maximum at week 10 (10.15% \u0026plusmn; 0.75), decrease by week 11 (8.68% \u0026plusmn; 0.54), R\u0026sup2; \u0026asymp; 0.8755.\u003c/p\u003e\u003cp\u003eBanana peel: rise until week 9 (9.29% \u0026plusmn; 0.63), followed by a decline to 7.30% \u0026plusmn; 0.58 at week 11, R\u0026sup2; \u0026asymp; 0.9402.\u003c/p\u003e\u003cp\u003eSucrose: nearly linear growth until week 10 (15.62% \u0026plusmn; 0.77), slight reduction by week 11 (13.30% \u0026plusmn; 1.37), R\u0026sup2; \u0026asymp; 0.9451.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStatistical analysis (ANOVA with Tukey\u0026rsquo;s post hoc test, p\u0026thinsp;\u0026le;\u0026thinsp;0.05) revealed that prickly pear peel differed significantly from orange peel, while no significant differences were observed among the other substrates.\u003c/p\u003e\u003cp\u003eSeveral factors likely account for the observed differences. The substrate composition is especially important: substrates containing both pulp and peel probably offer higher levels of simple sugars minerals, and moisture, promoting mycelial growth and biopolymer synthesis. Similarly, the availability of metabolizable carbon significantly influences polymer accumulation, with more accessible components promoting higher yields. These findings align with numerous studies that confirm agro-industrial residues such as fruit peels and crop processing byproducts are inexpensive, carbon-rich substrates for microbial biopolymer production [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, yields are highly dependent on factors like microbial strain, substrate pretreatment, and operational parameters including pH, temperature, and aeration [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond these, the physical characteristics of the substrates also play a critical role: more porous matrices enhance oxygen transfer and nutrient diffusion, whereas denser materials can restrict mass transfer and lower productivity. The stabilization or decline in yields after weeks 10\u0026ndash;11 may be associated with nutrient depletion, accumulation of inhibitory metabolites, or unfavorable pH shifts, factors that have been shown to negatively affect PHA accumulation in mixed microbial cultures [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA particularly noteworthy finding is that all biopolymers obtained in this study were extracellular, regardless of the substrate. During fungal growth, a thin polymer layer was consistently observed on the surface of the culture medium [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This extracellular localization offers significant technical advantages over intracellular polymer production: it eliminates the need for cell disruption and solvent-intensive recovery, simplifying downstream processing and reducing costs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These benefits are especially relevant when using low-cost agricultural residues, where minimizing operational costs is essential for ensuring economic feasibility [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhen compared with the literature, most studies using fruit peels, bagasse, and other agricultural byproducts report biopolymer yields rarely exceeding 10\u0026ndash;15% without prior substrate pretreatment or intensive process optimization [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. As highlighted in recent studies, the efficiency of microbial bioproduct generation from agroindustrial waste depends strongly on the substrate composition and fermentation method, particularly when using low-cost materials such as fruit residues. Therefore, the ~\u0026thinsp;17% yield obtained with the tejocote peel substrate in this study is particularly remarkable, as it exceeds most previously reported values and highlights week 9 as a key time point for process optimization.\u003c/p\u003e\u003cp\u003eIn summary, the data suggest that: Substrate selection critically affects biopolymer yields, extracellular production by \u003cem\u003eA. luteoalbus\u003c/em\u003e provides technical and economic advantages over intracellular systems, and further optimization of pH, substrate concentration, aeration, and agitation could potentially enhance productivity and product quality even more.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e displays scanning electron micrographs (SEM) taken at 500\u0026times; magnification, using an accelerating voltage of 15 kV and a working distance of 18 mm, with a JEOL JSM-6010LA microscope. The analysis was performed to assess the impact of substrate composition and fermentation time on the biopolymer's microstructure. The observed morphological features in these SEM images, including variations in surface arrangement, fiber formation, and density, are further summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, which correlates these microstructural changes with mechanical properties and yield outcomes for each substrate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe comparative analysis of how substrate type and fermentation time influence the microstructural development, mechanical properties, and yield of the resulting biopolymers is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Substrates rich in simple sugars, such as sucrose and tejocote peel pulp, promote the formation of uniform and durable films, while lignocellulosic residues like corncob and banana peel produce amorphous or brittle polymers with lower mechanical performance. These results confirm that substrate composition directly affects both the kinetics of polymer formation and the structural integrity of the material [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e Comparative Summary relationship between substrate type, fermentation time, microstructural evolution, mechanical properties, and polymer yields\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInitial Week\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInitial Microstructure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFinal Week\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFinal Microstructure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMechanical Properties\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYield\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOrange peel\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProtuberances, partial transformation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContinuous, uniform film\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh resistance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCorncob\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmorphous, poorly organized structure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSimilar morphology, no major changes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLow resistance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePrickly pear peel\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCompact film\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBrittle, fragmented structure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReduced integrity over time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTejocote peel pulp\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFibrous, incomplete transformation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContinuous, uniform film\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh resistance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBanana peel\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCompact film\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFibrous, brittle structure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReduced integrity over time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSucrose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 week\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eScaly, compact structure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eThin, resistant, flexible film\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh resistance \u0026amp; flexibility\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe comparative analysis in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that substrate type and fermentation time are crucial for the microstructural development, mechanical properties, and yield of biopolymers. Consistent with previous studies [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], substrates high in simple sugars, such as sucrose and tejocote peel pulp, support the quick formation of uniform, continuous, and mechanically strong films. In both cases, media containing readily metabolizable carbon sources led to faster polymer synthesis and better structural integrity of the final product, highlighting the significant impact of substrate composition on microbial biopolymer performance. This observation aligns with earlier reports indicating that the carbon source not only dictates microbial metabolic efficiency but also modulates polymer crystallinity and porosity, thereby determining its mechanical performance and potential functional applications [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCompared to thermally compressed synthetic polymers like High-Density Polyethylene (HDPE), which show a compact, uniform, and low-porosity structure associated with high mechanical strength and low degradability [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], polyhydroxyalkanoates (PHAs) display a more amorphous, porous, and irregular microstructure resulting from direct microbial biosynthesis without additional thermal processing [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This open structure, marked by interconnected cavities and uneven surfaces, is commonly reported in PHAs used in medical and agricultural applications [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Notably, the natural porosity of PHAs is linked to increased water absorption and molecular diffusion, properties that support controlled-release mechanisms in drug delivery and interactions with biological tissues [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, the microbial origin and biodegradable composition of PHAs enhance their environmental and physiological degradation compared to denser or more crystalline biopolymers. These combined attributes\u0026mdash;biodegradability, structural compatibility, and tunable porosity\u0026mdash;have driven their development in applications ranging from regenerative medicine and smart packaging to sustainable agricultural systems [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Physicochemical Properties\u003c/h2\u003e\u003cp\u003eThe results summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e demonstrate that all biopolymers obtained from different carbon sources exhibited similar physicochemical properties, including a dark brown, opaque appearance, brittle texture, and resistance to scratching. The melting point above 250\u0026deg;C indicates that the materials possess high thermal stability, a desirable feature for packaging and biomedical applications requiring dimensional stability at elevated temperatures.\u003c/p\u003e\u003cp\u003eThe combustion behavior, marked by a red flame, sweet odor during burning, and carbonization when removed from the flame, indicates a carbon-rich structure with high thermal resistance, aligning with findings for other fungal-derived biopolymers [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Moreover, the complete insolubility of all samples in both polar and non-polar solvents, including water, dimethyl sulfoxide, chloroform, and acetone, suggests a highly cross-linked or partially crystalline macromolecular network, like chitin and fungal melanins, which show comparable insolubility patterns [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Although this insolubility may limit some processing methods, it could provide benefits for water-resistant or barrier applications where solvent stability is advantageous.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental results of physicochemical tests performed using different carbon sources\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest performed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResult\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eInitial observations\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOpaque, plastic-like, brittle, resistant to scratching, dark brown color.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMelting point\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;250\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCombustion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIn flame: red color, sweet odor, remains ignited.\u003c/p\u003e\u003cp\u003eOutside flame: white color, pungent odor, with presence of ash.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSolubility\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInsoluble in water, dimethyl sulfoxide (DMSO), 2-isopropanol, toluene, hexane, xylene, carbon tetrachloride (CCl₄), chloroform, ethyl acetate, acetone.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e3.5 Elemental Analysis (CHNS) The elemental composition of the biopolymer obtained from the sucrose medium after eleven weeks is shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The polymer contained 44.5% carbon, 7.425% hydrogen, 1.80% nitrogen, and 0.605% sulfur, with the remainder likely consisting of oxygen and trace inorganic elements not measured by this method. The low nitrogen and sulfur content indicates a mainly carbohydrate- or polyphenolic-based composition rather than a protein-rich structure.\u003c/p\u003e\u003cp\u003eThe carbon fraction aligns with values reported for microbial polysaccharides and fungal cell wall polymers, supporting the hypothesis that the material comprises complex polysaccharides with both aromatic and aliphatic domains. Such compositions are typical for fungal exopolysaccharides, which frequently demonstrate thermal resilience and low solubility due to extensive hydrogen bonding and structural heterogeneity.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eElemental analysis results obtained with the Perkin Elmer 2400 Elemental Analyzer\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample amount analyzed (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e%\u003c/p\u003e\u003cp\u003eCarbon\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%\u003c/p\u003e\u003cp\u003eHydrogen\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003cp\u003eNitrogen\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%\u003c/p\u003e\u003cp\u003eSulfur\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.606\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.317\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;2)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e44.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Thermal Analysis (TGA and DSC)\u003c/h2\u003e\u003cp\u003eThe TGA thermogram (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) reveals three main stages of weight loss. The initial decrease below 120\u0026deg;C, corresponding to approximately 19.14% mass loss, reflects the evaporation of adsorbed and interlaminar water molecules, indicating a high moisture retention capacity within the polymeric network. The subsequent degradation phase between 250\u0026deg;C and 400\u0026deg;C shows three defined peaks at 286.20\u0026deg;C, 316.84\u0026deg;C, and 390.46\u0026deg;C, suggesting a stepwise breakdown of polysaccharidic components and associated organic moieties. This multistage behavior denotes structural heterogeneity and the coexistence of both thermolabile and thermoresistant domains within the matrix. Beyond 450\u0026deg;C, the mass loss rate decreases markedly, leaving a stable carbonaceous residue that points to the presence of aromatic or cross-linked fractions conferring enhanced thermal resilience to the biopolymer.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThermogravimetric analysis (TGA) results obtained with the Perkin Elmer TGA4000\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResult\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInitial sample mass (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.374\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaximum temperature \u0026ndash; 1st transition (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35.710\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMass loss \u0026ndash; 1st transition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6.211\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaximum temperature \u0026ndash; 2nd transition (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54.770\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMass loss \u0026ndash; 2nd transition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.929\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaximum temperature \u0026ndash; 3rd transition (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e286.200\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMass loss \u0026ndash; 3rd transition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.376\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaximum temperature \u0026ndash; 4th transition (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e316.840\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMass loss \u0026ndash; 4th transition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28.017\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaximum temperature \u0026ndash; 5th transition (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e390.460\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMass loss \u0026ndash; 5th transition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.241\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe DSC thermogram (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) further corroborates these findings. The glass transition temperature (Tg) at 197.52\u0026deg;C marks the transition from a rigid, brittle state to a soft, flexible material. The softening range (200\u0026ndash;250\u0026deg;C) preceded major degradation, indicating a narrow processing window for applications requiring thermal shaping or sterilization\u003c/p\u003e\u003cp\u003eBeyond 250\u0026deg;C, progressive endothermic events were observed at 416\u0026deg;C and 546\u0026deg;C, associated with structural decomposition rather than melting transitions. The absence of melting peaks suggests that the polymer either does not melt under the tested conditions (30\u0026ndash;550\u0026deg;C) or transitions directly from the solid to the degradation phase.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferential Scanning Calorimetry (DSC) results obtained with the Mettler Toledo DSC1\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1st transition (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2nd transition (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3rd transition (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4th transition (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5th transition (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6th transition (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOnset of melting\u003c/b\u003e\u0026thinsp;=\u0026thinsp;31.47\u0026deg;C\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeak melting temperature\u003c/b\u003e\u0026thinsp;=\u0026thinsp;63.20\u0026deg;C\u003c/p\u003e\u003cp\u003e\u003cb\u003eTwo-components temperature\u003c/b\u003e\u0026thinsp;=\u0026thinsp;123.82\u0026deg;C\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnthalpy\u003c/b\u003e = -260.07 J/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e84.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e197.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e274.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e416.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e546.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe high thermal stability, as demonstrated by onset degradation temperatures above 250\u0026deg;C and no melting transitions, indicates that this polymer could be suitable for biodegradable packaging materials, thermal insulation components, or biomedical membranes requiring dimensional stability under heat exposure.\u003c/p\u003e\u003cp\u003eThe sequential decomposition peaks suggest a heterogeneous macromolecular architecture, where different polysaccharide fractions or cross-linked domains degrade in stages, a phenomenon widely reported for microbial exopolysaccharides and lignocellulosic biopolymers [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, the insolubility across all tested solvents combined with thermal resilience implies a heavily hydrogen-bonded or partially crystalline structure, like fungal melanins or chitin derivatives, which exhibit remarkable chemical and thermal resistance and often require specialized processing routes for functionalization [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe effect of carbon source on polymer properties merits further investigation. Although sucrose produced the material analyzed here, fruit residues such as tecojote peel provided higher biopolymer yields in production trials; whether this also affects thermal or chemical properties should be explored in future studies.\u003c/p\u003e\u003cp\u003eFinally, despite the outstanding properties of the biopolymer (particularly its thermal stability and chemical resistance) it is essential to acknowledge several limitations that currently restrict its broader applicability. The material exhibited a brittle texture, indicating low flexibility and constraining its use in applications requiring elasticity or tensile strength, such as flexible packaging or biodegradable films.\u003c/p\u003e\u003cp\u003eAdditionally, the dark coloration observed may represent both an aesthetic and technological barrier for its incorporation into food packaging or biomedical applications where transparency and visual appearance are critical. Nevertheless, biopolymer bleaching strategies could potentially overcome this limitation.\u003c/p\u003e\u003cp\u003eAnother important constraint is its insolubility in all tested solvents, a feature that, while conferring stability under extreme conditions, significantly limits processing through conventional molding or extrusion techniques. Taken together, these findings suggest that, in its native form, the biopolymer is more suitable for specialized applications such as protective coatings, chemically resistant membranes, or barrier materials rather than as a direct substitute for mass-consumption plastics like polyethylene or polypropylene.\u003c/p\u003e\u003cp\u003eFurther research is required to address these limitations through chemical modifications, blending strategies, or advanced processing methods to enhance the material\u0026rsquo;s flexibility, transparency, and processability, thereby expanding its potential industrial applications.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of this study demonstrated that \u003cem\u003eA. luteoalbus\u003c/em\u003e is capable of producing an extracellular biopolymer using low-cost carbon sources, including agro-industrial residues, representing a sustainable strategy for the valorization of organic waste. The highest yield (~\u0026thinsp;17.10% \u0026plusmn; 1.29 at week 9 with pulp and tejocote peel) confirmed that substrate composition and incubation time are critical factors influencing the productivity of the process.\u003c/p\u003e\u003cp\u003ePhysicochemical, elemental, and thermal analyses showed that the obtained material exhibits high thermal stability, insolubility in common solvents, and resistance to degradation at temperatures above 250\u0026deg;C. These properties, together with its extracellular nature that simplifies recovery, position the biopolymer as a potential candidate for applications in biodegradable packaging, protective coatings, and biomedical materials, where chemical and thermal resistance are essential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003ePerspectives\u003c/h3\u003e\n\u003cp\u003eFuture work should focus on optimizing culture parameters such as pH, aeration, substrate concentration, and agitation conditions to further enhance productivity and evaluate process scalability in larger bioreactors.\u003c/p\u003e\n\u003cp\u003eAdditionally, it will be essential to conduct in-depth structural characterizationusing advanced spectroscopic (FTIR, NMR) and rheological techniques, as well as to assess mechanical properties\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003ebiodegradability\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eand performance under real-use conditions\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuch studies will help establish the technical, economic, and environmental feasibility of producing this biopolymer at an industrial scale, contributing to reducing the use of petroleum-derived plastics and promoting the development of a sustainable circular economy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions/Notes/Thanks/Other declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed equally to writing the article. Additionally, all authors have reviewed and approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes/Thanks/Other declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed equally to writing the article. Additionally, all authors have reviewed and approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Ramón Maruri-Gómez for language revision, Hugo Cuatecontzi-Flores, and Alejandra Sánchez-Barrera for their technical support. Likewise, the authors thank the PIAPIME ID 212312 FESC-UNAM. J Espinosa-Raya acknowledges the Instituto Politécnico Nacional and COFAA-SIP/IPN.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSchneiderman DK, Hillmyer MA (2017) 50th Anniversary Perspective: There Is a Great Future in Sustainable Polymers. Macromolecules 50:3733\u0026ndash;3749. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.macromol.7b00293\u003c/span\u003e\u003cspan address=\"10.1021/acs.macromol.7b00293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRitchie H, Samborska V, Roser M (2023) Plastic Pollution. 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Chemosphere 272:129884. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2021.129884\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2021.129884\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidad Nacional Autónoma de México, Facultad de Estudios Superiores Cuautitlán","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biopolymer production, Agro-industrial waste, Acrostalagmus luteoalbus, Sustainable materials, Waste-to-resource","lastPublishedDoi":"10.21203/rs.3.rs-7901410/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7901410/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e\u003c/em\u003e\u003cbr\u003e\nPlastic pollution has intensified the search for\u003cstrong\u003e \u003c/strong\u003ebiodegradable alternatives\u003cstrong\u003e \u003c/strong\u003efrom renewable sources. Microbial fermentation using\u003cstrong\u003e \u003c/strong\u003eagro-industrial residues\u003cstrong\u003e \u003c/strong\u003eoffers a sustainable strategy for producing biopolymers with reduced environmental impact. This study evaluated\u003cstrong\u003e \u003c/strong\u003ethe production and characterization\u003cstrong\u003e \u003c/strong\u003eof an\u003cstrong\u003e \u003c/strong\u003eextracellular biopolymer\u003cstrong\u003e \u003c/strong\u003esynthesized by \u003cem\u003eAcrostalagmus luteoalbus\u003c/em\u003e using low-cost carbon substrates.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u003cbr\u003e\n \u003c/strong\u003eFermentations were carried out for eleven weeks\u003cstrong\u003e \u003c/strong\u003eusing pulp with tejocote peel, fruit peels, and sucrose-based media\u003cstrong\u003e. \u003c/strong\u003eBiopolymer yields were quantified, followed by\u003cstrong\u003e \u003c/strong\u003ephysicochemical characterization\u003cstrong\u003e, \u003c/strong\u003eelemental analysis (CHNS)\u003cstrong\u003e, \u003c/strong\u003eand thermal assessments (TGA and DSC)\u003cstrong\u003e \u003c/strong\u003eto evaluate structural and functional properties.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u003cbr\u003e\n \u003c/strong\u003eAll substrates supported biopolymer synthesis, with\u003cstrong\u003e \u003c/strong\u003epulp with tejocote peel\u003cstrong\u003e \u003c/strong\u003eyielding the highest production (~17.10% ± 1.29 at week nine), indicating a strong influence of\u003cstrong\u003e \u003c/strong\u003esubstrate composition\u003cstrong\u003e \u003c/strong\u003eand incubation time\u003cstrong\u003e. \u003c/strong\u003eThe biopolymer was\u003cstrong\u003e \u003c/strong\u003edark brown, brittle, insoluble\u003cstrong\u003e \u003c/strong\u003ein polar and non-polar solvents, and\u003cstrong\u003e \u003c/strong\u003ethermally stable\u003cstrong\u003e, \u003c/strong\u003ewith degradation occurring above 250 °C\u003cstrong\u003e. \u003c/strong\u003eCHNS analysis showed a\u003cstrong\u003e \u003c/strong\u003ecarbon-rich, low-nitrogen composition\u003cstrong\u003e, \u003c/strong\u003ewhile TGA and DSC revealed\u003cstrong\u003e \u003c/strong\u003emulti-step degradation and\u003cstrong\u003e \u003c/strong\u003eno melting transitions\u003cstrong\u003e, \u003c/strong\u003esuggesting a\u003cstrong\u003e \u003c/strong\u003eheterogeneous, cross-linked polymeric network\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDiscussion:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u003cbr\u003e\n \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eextracellular nature\u003cstrong\u003e \u003c/strong\u003esimplifies recovery compared to intracellular polymers and combined with\u003cstrong\u003e \u003c/strong\u003ethermal stability\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esolvent resistance\u003cstrong\u003e, \u003c/strong\u003esupports applications in\u003cstrong\u003e \u003c/strong\u003ebiodegradable packaging, coatings, and biomedical materials.\u003cstrong\u003e \u003c/strong\u003eAgro-industrial residues represent a cost-effective\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esustainable carbon source\u003cstrong\u003e \u003c/strong\u003efor biopolymer production.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/em\u003e\u003cbr\u003e\n \u003cem\u003eAcrostalagmus luteoalbus\u003c/em\u003e offers a promising platform for producing\u003cstrong\u003e \u003c/strong\u003ebiodegradable, thermally stable biopolymers\u003cstrong\u003e \u003c/strong\u003efrom agro-industrial wastes, contributing\u003cstrong\u003e \u003c/strong\u003eto\u003cstrong\u003e \u003c/strong\u003ecircular economy strategies\u003cstrong\u003e \u003c/strong\u003eand industrial-scale sustainability efforts.\u003c/p\u003e","manuscriptTitle":"Extracellular Biopolymer Production by Acrostalagmus luteoalbusfrom Agro-Industrial Wastes: Toward Sustainable Material Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 05:52:35","doi":"10.21203/rs.3.rs-7901410/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26bcd278-3c1f-4415-91da-48ea43ab37c2","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56555800,"name":"Biopolymers"},{"id":56555801,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2025-10-22T05:52:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-22 05:52:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7901410","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7901410","identity":"rs-7901410","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}